Proc. Natl. Acad. Sci. USA Vol. 92, pp. 2894-2898, March 1995 Pharmacology

Combinatorial diffusion assay used to identify topically active melanocyte-stimulating hormone receptor antagonists (synthetic combinatorial chemistry/Xenopus laevis/skin tone) J. MARK QUILLAN*, CHANNA K. JAYAWICKREMEt, AND MICHAEL R. LERNER*t Departments of *Pharmacology and tInternal Medicine, Boyer Center for Molecular Medicine, Yale University School of Medicine, P.O. Box 9812, New Haven, Connecticut 06536-0812 Communicated by Aaron B. Lerner, Yale University, New Haven, CT, November 30, 1994

ABSTRACT a-Melanocyte-stimulating hormone (av- hypothesectomy for treatment of metastatic breast cancer (19). MSH) is implicated in pigmentation, central nervous system However, the complex and far-reaching physiological interac- and immune system functions, growth, mitogenisis, and mel- tions of the pituitary obscure demonstration of a direct anoma. Evaluation ofthese roles has been hindered by the lack involvement of a-MSH in tonic melaninzation and outward of a-MSH antagonists. A combinatorial chemistry-based dif- appearance. The possible involvement of extrapituitary MSH fusion assay is used to find random tripeptides that antago- released in humans by dermal keratinocytes after exposure to nize normal frog and human melanoma MSH receptors and ultraviolet light (20) also has not yet been examined. Identi- to identify pharmacological groups responsible for receptor fication of MSH antagonists in this report, antagonists effec- interaction. The a-MSH antagonist D-Trp-Arg-Leu-NH2 is tive at the human receptor, opens the way to address these and used to demonstrate directly the contribution of MSH to other questions concerning the importance and various phys- normal skin tone in frogs following injection or topical iological roles of the tridecapeptide a-MSH. application. MATERIALS AND METHODS a-Melanocyte-stimulating hormone (a-MSH) is potentially Library Construction and Synthesis. Diffusion assays (de- involved in numerous important physiological and patholog- scribed below) were used to identify MSH receptor antagonists ical processes ranging from pigmentation to melanoma (1-9). by randomly screening a synthetic combinatorial library con- These roles have not been closely examined, in part at least taining 221,184 sequence combinations (Fig. 1 Upper). The because of a lack of MSH receptor antagonists. Preliminary library was divided into 96 sublibraries of 2304 (1 x 48 x 48) reports of compounds that inhibit a-MSH-induced darkening sequence combinations each, based on the chemical structure in Rana pipiens skin assays have been made (10), but it is of the amino-terminal position. Library synthesis was carried unclear how receptor specific these compounds are or if they out essentially as described for multiuse peptide libraries (11) are effective in blocking responses to MSH in other species or by using fluorenylmethoxycarbonyl (Fmoc) chemistry on in vivo. One method typically used to identify antagonists to 4-methylbenzhydrylamine (MBHA)-linked polystyrene resin peptide receptors has been to manipulate the size and se- in combination with a standard simultaneous multiple peptide quence of the native peptide hormone. Another method has synthesis protocol (21, 22). Briefly, 0.5 mmol of derivatized relied on random screening of thousands ofcompounds. In this amino acid was dissolved in 1.25 ml of N-methylpyrrolidone report we describe the use of a combinatorial diffusion assay, (NMP)/1.0 ml of N,N-dimethylformamide containing 0.45 M an assay that separates molecules in time and space (11), to O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexa- randomly search for tripeptide and tripeptide-like molecules fluorophosphate (HBTU) and 0.45 M 1-hydroxybenzotriazole that antagonize the ca-MSH receptor. The small and uncom- (HOBT). This mixture was then added to the resin, followed plicated structure of these peptide antagonists provides useful by 1.0 mmol of N,N-diisopropylethylamine and allowed to structural information for determining receptor interactions. react for 30 min at 25°C. The resin was then filtered and rinsed Pigmentary phenomena serve as an example of the many with NMP, and coupling efficiency was verified by the ninhy- areas of study that stand to benefit from the development of drin test (23). The resin was deprotected with 20% piperidine specific MSH receptor antagonists. Pigmentation has long in NMP, filtered, and rinsed with NMP. Side-chain protection been recognized for playing a role in camouflage and sun groups were removed by mixing with cleaving reagent (90% protection, but the extent of a-MSH involvement in regulation CF3COOH/5% thioanisole/2.5% ethanedithiol/2.5% water, of basal skin tone is not yet firmly established. It is well known vol/vol) at 25°C for 1 hr. The cleaving reagent was removed by that injection of ca-MSH into a variety of animals including filtration, and beads were washed sequentially with dichlo- humans causes an increase in skin tone beyond basal levels (1, romethane, NMP, and methanol, before drying under vacuum. 2), and there is evidence in some species that plasma a-MSH Preparation of Library Beads for Screening. In preparation may correlate with changes in background and animal color for screening, beads were spread over the surface of a trans- (12-14), but these do not provide conclusive information with parent polyethylene film (Glad ClingWrap), and -4% of the regard to the extent of MSH involvement in the setting or total bead-attached molecules were released by using a con- maintenance of basal tone. Support for the possible involve- trolled gas-phase CF3COOH cleavage procedure (100% gas- ment of a-MSH in basal skin tone comes primarily from eous CF3COOH at 20°C and 20-33 kPa for 10 hr). Beads were experiments in which removal of the pituitary gland induces detoxified by neutralizing CF3COOH salts with gaseous NH3/ pallor in fish, amphibians, and lower mammals (15-18) and H20 (vaporized 30% ammonium hydroxide solution; J. T. from anecdotal clinical reports, given without explanation or kPa 1 These speculation, that humans become abnormally pale following Baker, Phillipsburg, NJ) at 20°C and 20-33 for hr. the loss of pituitary function due to disease or following Abbreviations: MSH, melanocyte-stimulating hormone; Abu, 2-ami- nobutyric acid; y-Abu, 4-aminobutyric acid; e-Ahx, 6-aminohexanoic The publication costs of this article were defrayed in part by page charge acid; Aib, 2-aminoisobutyric acid; ,8-Ala, 3-aminopropionic acid; Orn, payment. This article must therefore be hereby marked "advertisement" in ornithine; Hyp, trans-hydroxyproline; Nle, norleucine; Nva, norvaline; accordance with 18 U.S.C. §1734 solely to indicate this fact. MBHA, 4-methylbenzhydrylamine; AVT, [Arg8]vasotocin. 2894 Pharmacology: Quillan et al. Proc. NatL Acad Sci. USA 92 (1995) 2895 Position Table 1. IC50 values for selected sequences 1 2 3 Peptide Sequence IC50, ,uM AA1 AAt A D-Trp-Arg-Xaa-NH2 Al Leu 0.62 ± 0.15 A2 Nle 0.93 ± 0.22 A3 Nva 3.3 ± 1.1 AA37 AA37 A4 Met 5.6 ± 2.6 A5 D-Nle 9.9 ± 1.8 Abu Abu A6 Ile 49 ±9 596 ?Abu -tAbu NH2 e-Ahx s-Ahx A7 Abu 82 ±41 Aib Aib A8 Val 237 ± 100 3-Ala f-Ala A9 Arg 261 ± 68. Hyp Hyp A10 D-Arg 664 ± 397 Nie Nbe MNIe D-Nle All y-Abu Inactive Nva Nva A12 s-Ahx2 Inactive D-Nva D-Nva A13 Ala Inactive Orn Orn A14 3-Ala Inactive B D-Trp-Xaa-Nle-NH2 Bi Lys 15 ± 1 B2 D-Arg 30 ± 11 B2 Leu 48 ±2.2 B4 Nle 59 ± 12 B5 Ala 65 ± 18 B6 Met 121 ± 27 B7 Abu 405 ± 69 B8 Asp Inactive C Xaa-Arg-Nle-NH2 Cl D-Phe 4.4 ± 1.2 C2 D-Tyr 28 ± 2 C3 Ac-D-Trp 43 ± 8 FIG. 1. (Upper) The synthetic peptide combinatorial library used to C4 Trp 100 ± 2 screen for and study MSH receptor antagonists was composed of 96 CS D-His 318 ± 105 sublibraries. Z96 is used to represent the unique amino terminus position 1 molecule that comprises the 96 sublibraries. Position 1 is ophore cell layer by an intervening 2-mm thickness of agarose, composed of 1 of the 48 molecules listed for positions 2 and 3, or 1 of allowing only dissociated free molecules to interact at the cell 48 acetylated equivalents. AAl to AA37 at positions 2 and 3 are the 20 standard L-amino acids (not prefixed) minus cysteine plus 18 surface. Responses were observed with computervideo images equivalent dextrorotatory (D) isoforms; 11 additional molecules, as (TLC-Image software from Biological Detection Systems, listed, bring the total to 48. Abu, 2-aminobutyric acid; a-Abu, 4-ami- Pittsburgh) generated by a high-resolution charge-coupled nobutyric acid; E-Ahx, 6-aminohexanoic acid; Aib, 2-aminoisobutyric device camera (Videc MEGAPLUS model 1400 from Kodak). acid; 3-Ala, 3-aminopropionic acid; Hyp, trans-hydroxyproline; Nle, Dose-Response Studies. Peptides for dose-response studies norleucine; Nva, norvaline; Orn, ornithine. (Lower) Video image of were synthesized on Rink amide MBHA-resin (Novabiochem) a-MSH antagonist-like responses produced by beads from the com- by using standard fluorenylmethoxycarbonyl chemistry (see binatorial sublibrary containing D-Trp-l. (Left) Image showing a 6-cm ref. and were SMART culture dish of agarose-covered Xenopus melanophores (pretreated 11) purified by HPLC (Pharmacia with 1 nM melatonin for 30 min and 15 nM a-MSH for 2 min) just after system Sephasil C18 SC 2.1/10 reverse-phase column) with a the application of "600 beads from the D-Trp-l sublibrary. Melano- linear gradient of solvent A (0.1% CF3COOH in water) to phore cells aggregate intracellular pigment granules in response to solvent B (0.085% CF3COOH in acetonitrile). Peptides were melatonin and disperse pigment once a-MSH is added. (Right) After eluted at 180 ml/min with a gradient of 10-40% solvent B over 60 min, white circular patterns appeared in response to release from 20 min. Dose-response measurements were quantitated by nearby beads; patterns are consistent with blockade of the MSH measuring transmittance through a monolayer of cultured receptor. Circle diameters increase over time as active molecules melanophores pretreated with 1 nM melatonin (see ref. 25), diffuse from their bead of origin through the gel matrix, allowing more cells to aggregate their pigment. and curves were fit with a logistic equation (26). Animals and in Vivo Treatments. Healthyjuvenile and adult techniques allow the detached synthetic molecule to remain Xenopus laevis were used for injection and topical drug appli- noncovalently associated with its bead of origin until released cations. Animals were maintained in a 10% (vol/vol) Ringer's into assay. The kinetics of bond cleavage from MBHA-linked solution (11.5 mM NaCl/0.26 mM KCl/0.2 mM CaCl2/1.0 mM resin has been described elsewhere (see ref. 11). The 10 pmol Hepes, pH 7.6) on a black background in the dark ("dark (or 4%) release level per bead was used so that responses from black-adapted") or in a normal 24-hr night/daylight cycle on molecules with potencies as low as 100 ,uM could be observed. a white background ("light white-adapted") or a black back- Diffusion Assays. Cultured Xenopus laevis melanophores ground ("light black-adapted"). Subcutaneous injections (see (24), 1.5 x 106 cells, were seeded into a 6-cm culture dish Fig. SLeft) were performed by using approximately 0.25 ml of (Falcon) and maintained for -72 hr at 27°C in fibroblast- peptide dissolved in buffer (142 mM NaCl/4.4 mM KCl/1.1 conditioned medium. Cells were exposed to 1 nM melatonin mM CaCl2/1.2 mM NaHCO3/1.0 mM MgCl2/2.8 mM dex- for 30 min prior to replacement of the medium with 5 ml of a trose). Injected animals were kept in a humid, darkened 1% (wt/wt) agarose (SeaPlaque from FMC Corporation) with environment for 20 min and reintroduced to their water culture medium mixture containing 1 nM melatonin and 15 habitat after the drug had taken effect. Topical applications nM a-MSH. After 2 min, the agarose was overlaid (face down) were performed with peptides dissolved water and applied to with polyethylene film containing a layer of prepared combi- the skin surface with lens paper (American Scientific Products, natorial beads. The beads remain separated from the melan- McGaw Park, IL). 2896 Pharmacology: Quillan et al. Proc. NatL Acad Sci. USA 92 (1995) cAMP Measurements. Intracellular cAMP was quantitated detection of multiple antagonist signals arising from various in cultured Xenopus melanophores grown to confluency in sublibraries provided an opportunity to compare similarities 24-well tissue culture plates (Falcon) or in transfectedXenopus and differences in antagonist structure. This information is fibroblast cells (24) by measuring displacement of potentially useful in making determinations of the molecular [8-3H]cAMP from cAMP-binding protein (27) with a components that contribute to receptor interaction and block- [8-3H]cAMP kit obtained from Amersham. Intracellular ade and may even be used toward development of nonpeptide cAMP was extracted with 1 ml of 60% (vol/vol) ethanol and antagonists. centrifuged to remove cellular debris, and 400-,ul aliquots were Identification of active molecules was carried out by itera- lyophilized for quantitation. Transfections were performed by tively screening subpools of the D-Trp-1 sublibrary. Response electroporation (5 x 10-6 cells per 400 ,ul in 96 mM NaCl/1.9 patterns generated in these experiments suggest that the most mM KCl/1.0 mM kH2 p04/6.6 mM Na2H P04, pH 7.0, potent signals emanate from subpools containing Arg at containing 10 jig of test cDNA) in 0.2-cm cuvettes in a BTX position 2. Subpools containing Lys-2, D-Arg-2, Met-2, Leu-2, ECM-600 gene-transfer apparatus at 475 V, 720 ohm, and 400 or Nle-2 also produced positive, although weaker, antagonist- ,uF; 48 hr after transfection, fibroblast cells plated to conflu- like signals. In Table 1, peptide sequences A1-A6 show the six ency in 12-well tissue culture plates (Falcon) were rinsed for most potent molecules identified by screening the D-Trp-Arg- 1 hr with 70% (vol/vol) L-15 medium (Sigma) containing 0.5% Xaa subpool. bovine serum albumin and again for 5 min with added 0.5 mM After sequence identification, candidate antagonists were 3-isobutyl-1-methylxanthine (IBMX; Aldrich). resynthesized, purified, and tested (Fig. 2). The two most potent antagonists, D-Trp-Arg-Leu-NH2 and D-Trp-Arg-Nle- RESULTS AND DISCUSSION NH2, have IC50 values (mean ± SEM) of 620 ± 150 nM and Random screening of the combinatorial library illustrated in 930 ± 220 nM, respectively, against 15 nM a-MSH. These Fig. 1 indicated the presence ofmultiple antagonist-like signals antagonists are competitive and specifically block a-MSH- from sublibraries containing D-Trp, D-Phe, D-Tyr, and Ac-D- induced activation of endogenous amphibian and transfected Trp at amino-terminal position 1; we use the amino acid human MSH receptors (Figs. 2, 3, and 4). D-Trp-Arg-Leu-NH2 three-letter code when referring to sequence positions. The has an equilibrium Kd of 63 ± 15 nM (Fig. 2 Center), in line with most numerous and potent signals were produced by beads a predicted IC50 of -96 nM had a-MSH been applied at its from the D-Trp-1 sublibrary. These responses, seen in the EC50 value. presence of 15 nM a-MSH (Peninsula Laboratories; EC50 2 The multiple MSH receptor antagonist molecules identified nM), were not visible when 8 nM [Arg8]vasotocin (AVT, from the D-Trp-Arg-Xaa subpool constitute a set of structur- Sigma; EC50 2 nM) or 4 nM vasoactive intestinal peptide ally related analogs (Fig. 5), a set of "permissible" substitu- (Sigma; EC50 1 nM) was used to activate pigment dispersion tions, in that receptor activity is not abolished by differences in place of a-MSH. in structure at position 3. Within this group, IC measurements By screening large numbers of random small tripeptide-like reveal that antagonist potency is positively correlated with the molecules, it was possible to bypass the more traditional length of the hydrocarbon side-chain group (i.e., approach to peptide antagonist development that involves Nle>Nva>Abu>Ala) and negatively correlated with the pres- manipulation of known ligand structures, an approach that ence of a ,-methyl group (i.e., Nva>Ile and Abu>Val). often leads to substantially larger molecules. In addition to Removal of the -y-methyl group from Leu-3 results in a 50% finding antagonists with molecular masses of <500 Da, an decrease in antagonist potency as evidenced by comparison to attractive start for molecular modeling, the simultaneous Nva-3. When Met is located at position 3, there is a 6-fold

100 4 0.6

0 75 0 3 2 0.4 50 E l 2 I-, I- E 0 0 25 0.2 1

0 0 0.0 -8 -7 -6 -5 -4 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 Antagonist, log M dWRL, log M a-MSH, log M

FIG. 2. Peptides that reverse ca-MSH-induced pigment darkening in diffusion assays are tested in vitro. (Left) Candidate antagonists are tested for their ability to independently inhibit a-MSH-induced pigment dispersion. The graph shows an example of inhibition curves for three peptides Al, A4, and A6 (see Table 1) identified from diffusion assays. Two other molecules not found in the library screens, A9 and All, are included for comparison. Each point represents the mean and sample SD of four independent measurements taken 2 hr after addition of 15 nM a-MSH plus the indicated concentration of test antagonist. Results are expressed as a percentage relative to treatment with a-MSH alone. (Center) Competitive inhibition of a-MSH by D-Trp-Arg-Leu-NH2 (dWRL) is demonstrated by using Schild regression analysis (28). The negative logarithm of the equilibrium Kd is 7.2 ± 0.1, and the slope of the regression is 1.06 ± 0.03. Broken lines indicate 99% confidence level. dr, dose ratio. (Right) Dose ratios (dr) for Center were obtained from concentration-response curves for a-MSH taken in the absence (0) and presence (*) of 1 ,uM, 10 ,uM (A), and 100 ,LM (0) dWRL. ECso for a-MSH alone is 2.5 ± 0.3 nM and for a-MSH + dWRL is 4.8 ± 0.6 ,uM. Each point represents the mean and sample SD of four independent transmittance measurements. Ti = initial transmittance (2 min); Tf = final transmittance (60 min). dWRL causes no change in EC5o values for either vasoactive intestinal peptide or AVT (data not shown). Pharmacology: Quillan et al. Proc. NatL Acad Sci. USA 92 (1995) 2897 AA R-Group ICSO (PK) 3 z 32 r 2 Leu 10 ysp 0.6 ± 0.2 Nle etvep-1*p 0.9 ± 0.2 '4 24 Nva p,-%%p 3.3 ± 1.1 0 E Met 0 p 5.6 ± 2.6 16 '4 49±9 0 nIe P 8 Abu P* 82±41

Val 0P 237 ±100 O L Ala *--ftp Inactive FIG. 3. D-Trp-Arg-Nle-NH2 (dW-R-Nle), at 40 ,uM, blocks 10 nM a-MSH-mediated cAMP second-messenger stimulation in cultured P* Inactive Xenopus melanophores but does not block cAMP stimulation evoked PAIa by 8 nM AVT. Oxytocin antagonist GVT {[Pmp , Tyr(Me)2, FIG. 5. Structure-function comparisons at the third position for Orn8]vasotocin from Peninsula Laboratories, where Pmp = 1-(,B- the six most potent L amino acids identified by random screening from mercapto-,B,l-cyclopentamethylene)propionic acid} at 20 AM is used subpools containing D-Trp at position 1 and Arg at position 2 (see Fig. as a control to block responses evoked by 8 nM AVT. Each bar 1 Upper). Three additional substitutions at position 3 not found in the represents the mean and sample SD of four independent measure- random screen are included for comparison. Antagonist activity ments. *t test, P < 0.001, for all groups except those bearing asterisks. correlates with hydrophobicity and charge characteristics of the R group found at position 3. Chain length and steric hindrance by reduction in antagonist potency compared to Nle-3 (which has 3-carbon substitution are also important. a similar but non-sulfur-containing R group). The strongest hydrophilic, charged substitution tested, Arg-3, causes a 300- sequences). D-isomer subpools also contained positive signals fold decrease in potency compared with Nle-3 (see Table 1, A but produced weaker signals than the L-isomer subpools. D-Nle-3, included for comparison, was 1/10th as potent as the

-j L-isomer position 3. The vast majority of tripeptides screened with the diffusion 5 a 20 assay show no a-MSH antagonist-like activity. Although this is .x + expected, given the high specificity of receptor-ligand inter- Ik actions, negative results are informative. The lack of activity in most library pools tested suggests that changes made to 100 U) IL o .bL position 1 are not well tolerated in terms of receptor interac- 80 [cI tion. Besides D-Trp-1, only the clearly related D-Phe-1 and, to 60 ML a lesser extent, D-Tyr-1 in the nonacetylated pools were found to display significant a-MSH antagonist-like activity. Confir- mation ofthis observation comes from the finding that they are E also the two most potent permissible position 1 substitutions in a general Xaa-Arg-Nle-NH2 structure (see Table 1, peptides Cl and C2), where Xaa represents all 48 nonacetylated 4 combinations described in Fig. 1. Thus, positive signals ob- 10 served in screening subpools probably arise as a result of structural similarity to positives in the D-Trp-1 library and do not represent additional unrelated structures. A similar picture 'a apparently holds for positives observed within the position 2 0 subpools of the D-Trp-1 library. Replacements of Xaa-2 in a 0 D-Trp-Xaa-Nle-NH2 structure with Lys, D-Arg, Met, Leu, and C= 0 Nle result in analogs that display antagonist activity (see Table hMel MSHR I Vector alone 1, peptides Bi to B5), and, as expected, each has a potency less FIG. 4. Functional antagonism of a human MSH receptor by than when Arg occupies position 2. Therefore, it appears that D-Trp-Arg-Leu-NH2 (dWRL) is demonstrated in Xenopus fibroblasts molecules selected from random screens because they give the (26) transfected with "Vector alone" (pcDNAI/NEO; Invitrogen) or strongest signals are in fact the most potent MSH-receptor with "hMel MSHR" (pcDNAI/NEO containing a human melanoma antagonists present in the library. Variations in the combina- MSH receptor insert; gift from Roger Cone of the Vollum Institute, torial assay (bead size, for example) contribute less to differ- Portland, OR; see ref. 29). Control = no additional drugs. MSH = 5 ences in signal strength than does a 1 or 2 order of magnitude nM a-MSH. dWRL concentration is 10 ,uM. Forskolin = 100 ,uM difference in potency. 7(3-deacetyl-713-(y-N-methylpiperazino)butyrylforskolin from Calbio- To begin to assess the possible contribution of a-MSH to chem). Test drugs were added with 3-isobutyl-1-methylxanthine tonic skin tone, dark black-adapted Xenopus laevis were in- present for 45 min, and intracellular cAMP was extracted with 1 ml of 60% ethanol per well. Each bar represents the mean and SSD of three jected with D-Trp-Arg-Leu-NH2 (40 ,umol/kg) or with D-Trp- independent measurements, except for the control-HMel MSHR group, Abu-Arg-NH2 (control). D-Trp-Arg-Leu-NH2 caused com- where n = 6. *By the t test, P < 0.006 for all groups except other groups plete pallor in every frog tested (n = 10) within 20 min (see Fig. bearing a single asterisk. **By the t test, P < 0.006 for all other groups. 6 Left), whereas no change was observed in the control group 2898 Pharmacology: Quillan et al. Proc. NatL Acad ScL USA 92 (1995)

FIG. 6. MSH antagonists isolated from diffusion assays are active in vivo. (Left) D-Trp-Arg-Leu-NH2 (dWRL; 40 ,umols/kg) induces pallor when injected into Xenopus (white animals), while injection of a control peptide (D-Trp-Abu-Arg-NH2) at the same concentration causes no response (dark animals). (Right) Topical application of the tripeptide dWRL (1 mM in H20) to the skin of Xenopus laevis causes a local lightening in pigmentation. (n = 10). Similar results were observed with light black-adapted Xenopus. (n = 6). To demonstrate that antagonist lightening is 7. Strand, F. L., Zuccarelli, L. A., Williams, K. A., Lee, S. J., Lee, induced locally at the level of the skin, D-Trp-Arg-Leu-NH2 was T. S., Antonawich, F. J. & Alves, S. E. (1993)Ann. N.Y Acad. Sci. applied topically (1 mM in H20) to the skin surface of fully 680, 29-49. 8. Halaban, R., Tyrrell, L., Longley, J., Yarden, Y. & Rubin, J. conscious, nonpretreated Xenopus (Fig. 6 Right). These results (1993) Ann. N.Y Acad. Sci. 689, 290-300. indicate that the peptide acts transdermally in vivo and is clearly 9. Varga, J. M., Dipasquale, A., Pawelek, J., McGuire, J. S. & visible in both black-adapted animals and in white-adapted Lerner, A. B. (1974) Proc. Natl. Acad. Sci. USA 71, 1590-1593. animals (as shown in Fig. 6 Right) placed on a black background 10. Al-Obeidi, F., Hruby, V. J., Hadley, M. E., Sawyer, T. K & 2 hr prior to topical application. It appears, therefore, that tonic Castrucci, A. M. D. L. (1990) Int. J. Pept. Protein Res. 35, 228- coloration is indeed mediated by endogenous melanotropin and 234. that removal of this influence causes the frog to assume a 11. Jayawickreme, C. K., Graminski, G. F., Quillan, J. M. & Lerner, lightened "albino-like" state. The effects of injection and topical M. R. (1994) Proc. Natl. Acad. Sci. USA 91, 1614-1618. 12. Wilson, J. F. & Morgan, M. A. (1979) Gen. Comp. Endocrinol. 38, application were reversible after several hours and, in a few cases 172-182. of repeated or concentrated applications, after a few days, upon 13. Bowley, T. J., Rance, T. A. & Baker, B. I. (1983) J. Endocrinol. reintroduction of the frogs to their water habitat. 97, 267-275. These experiments demonstrate the usefulness of diffusion 14. Rodrigues, K. T. & Sumpter, J. P. (1984) J. Endocrinol. 101, assays for the discovery of novel drugs and for identification 277-284. of important pharmacological groups involved in receptor 15. Allen, B. M. (1916) Science 44, 755-758. interaction. And since the antagonists identified in this report 16. Smith, P. E. (1916) Science 44, 280-282. are effective at the human melanocyte a-MSH receptor, it 17. Rust, C. C. (1965) Gen. Comp. Endocrinol. 5, 222-231. 18. Chavin, W. (1956) J. Exp. Zool. 133, 1-36. should be of interest to assess any possible contribution of the 19. Felig, P., Baxter, J. D., Broadus, A. E. & Frohman, L. A., eds. synergistic mitogen a-MSH-be it from the pituitary or (1987) Endocrinology andMetabolism (McGraw-Hill, New York), keratinocytes (20)-to the pathobiology of melanoma and to 2nd Ed., p. 26. learn how a-MSH is involved in normal baseline pigmentation 20. Luger, T. A., Schauer, E., Trautinger, F., Krutmann, J., Ansel, J., in mammals. D-Trp-Arg-Leu-NH2 or its related tripeptide or Schwarz, A. & Schwarz, T. (1993) Ann. N.Y Acad. Sci. 680, tripeptide-like molecules may also be useful for addressing 567-570. other issues concerning the widespread effects of MSH 21. Lam, K S., Salmon, S. E., Hersh, E. M., Hruby, V. J., Kazmier- throughout the body and in the possible roles of the multiple ski, W. M. & Knapp, R. J. (1991) Nature (London) 354, 82-84. 22. Houghten, R. A., Pinilla, C., Blondelle, S. E., Appel, J. R., MSH receptor subtypes present in the central nervous system. Dooley, C. T. & Cuervo, J. H. (1991) Nature (London) 354, We thank Kristine Harris for her excellent assistance with peptide 84-86. synthesis and Patti Broccoli for her assistance in preparation of the 23. Sarin, V. K, Kent, S. B. H., Tam, J. P. & Merrifield, R. B. (1981) manuscript. Anal. Biochem. 117, 147-157. 24. Daniolos, A., Lerner, A. B. & Lerner, M. R. (1990) Pigm. Cell 1. Eberle, A. N. (1988) The Melanotropins: Chemistry, Physiology Res. 3, 38-43. and Mechanism ofAction (Karger, Basel). 25. Potenza, M. N., Graminski, G. F. & Lerner, M. R. (1992) Anal. 2. Lerner, A. B. & McGuire, J. S. (1961) Nature (London) 189, Biochem. 206, 315-322. 176-179. 26. De Lean, A., Munson, P. J. & Rodbard, D. (1978) Am. J. Physiol. 3. Murphy, M. T., Richards, D. B. & Lipton, J. M. (1983) Science 235, E97-E102. 221, 192-193. 27. Gilman, A. G. (1970) Proc. Natl. Acad. Sci. USA 67, 305-312. 4. Cannon, J. G., Tatro, J. B., Reichlin, S. & Dinarello, C. A. (1986) 28. Arunlakshana, 0. & Schild, H. 0. (1959) Br. J. Pharmacol. 14, J. Immunol. 137, 2232-2236. 48-58. 5. Tatro, J. B. (1990) Brain Res. 536, 124-132. 29. Mountjoy, K. G., Robbins, L. S., Mortrud, M. T. & Cone, R. D. 6. De Weid, D. (1993) Ann. N.Y Acad. Sci. 680, 20-28. (1992) Science 257, 1248-1251.